Screening For Novel Esterases From Metagenomic Library And Its Catalytic Performances In Non-Aqueous Media

The natural environment microorganisms widely distributed variety, it is rich in genetic resources, but 99% of the microorganisms in the present experimental conditions are not easy to obtain pure cultures. Metagenomics, which is a cultivation-independent method, can be used to avoid this inherent loss of diversity and is regarded as one of the most powerful approaches to investigate the potential of particular microorganisms without the need for culturing. Esterases as a general class of carboxylic ester hydrolases are widely used in different research fields. It is with high theoretical and practical application value, which showing great potential in biocatalysis. In contrast to lipases, only a few esterases have practical use in organic synthesis. Thus, by exploiting microbial rich resource to develop novel and excellent characteristics of esterase, has very important practical significance and industrial application values.Based on the above purpose, the present study is to develop novel esterase gene. A metagenomic library was constructed using the total genomic DNA extracted from marine mud. A novel esterase EST4 with high activity and high enantioselectivity was screened. Based on the purpose of the development and practical application, EST4 was successfully used in a non-aqueous media catalytic kinetic resolution and synthesis of important industrial acetic acid esters. In addition, by immobilization technology, the immobilized EST4 has remarkable stability and more efficient. Unique nature and excellent catalytic effect esterase, EST4 proved to be a very promising biocatalyst, suitable for large-scale commercial production. The results obtained are as follows:(1) The DNA was extracted directly from the marine mud and used to construct a metagenomic fosmid library, which capacity of about 40,000 clones. Based on functional heterologous expression,34 clones that showed lipolytic activity were isolated. The five clones with the largest halos were identified, and the corresponding genes were successfully overexpressed in Escherichia coli. Molecular analysis revealed that these encoded proteins showed 48-79% similarity with other proteins in the GenBank database. Multiple sequence alignment and phylogenetic tree analysis classified these five protein sequences as new members of known families of bacterial lipolytic enzymes (Family IV, Family V and Family VIII). These new esterases provide materials for subsequent experiments.(2) The five new esterase genes were successfully cloned and expressed in E. coli in soluble. Among the five esterases, EST4 was studied in further detail owing to its strong hydrolytic activity toward tributyrin. Characterization of EST4 indicated that it is an alkaline esterase that exhibits highest hydrolytic activity towards p-nitrophenyl butyrate (specific activity:1389 U mg-1) at 45 ℃ and pH 8.0. The half-life of EST4 is 55 and 46 h at 40 and 45 ℃, respectively, indicating a relatively high thermostability. EST4 also showed remarkable stability in organic solvents, retaining 90% of its initial activity when incubated for 12 h in the presence of hydrophobic alkanes. The surfactants and metal ions did not show any significant stimulation of the enzyme activity. Enzymatic properties of EST4 provide a certain foundation for industrial applications. In particular, the high organic solvent tolerance and thermostability of EST4 were very favorable in non-aqueous media, which laid a good foundation for the subsequent catalytic reaction.(3) Optically pure secondary alcohols as an important class of chiral intermediates, were the production of important drugs chiral building blocks and can be transformed into important bioactive molecules. Esterase-catalyzed kinetic resolution of various racemic alcohols via enantioselective hydrolysis and acylation is considered to be an effective way. Using a-phenethyl alcohol as a model substrate, the effects of various optimization parameters on the kinetic resolution catalyzed by the EST4 whole-cell biocatalyst were investigated and optimized. The best optimized reaction parameters to obtain the good conversion and enantioselectivity are as: molar ratio of a-phenylethanol/vinyl butyrate,1:3, at 30 ℃ in n-hexane (log P=3.5). EST4 achieved maximum catalytic activity can be obtained while alcohol substrate is greater than 99.9% ee with E>200. EST4 had a relatively broad substrate spectrum and showed excellent enantioselectivity (up to 99.9% ee) for the synthesis of a series of enantiopure alcohols based on an optimized efficient kinetic resolution system. The E values for half of substrates were above 200. Meanwhile, EST4 also exhibited strong tolerance against high substrate concentrations up to 1 M, and the optical purity of the desired secondary alcohols was kept above 99% ee.(4) Acetate ester compounds have a great application mainly in food and cosmetic industries due to their characteristic fragrance and flavor. The lyophilized E. coli as a whole-cell biocatalyst was for the synthesis of acetate ester compounds. EST4 as whole-cell catalysts in the non-aqueous media synthesis acetate optimum conditions were:n-hexane as the reaction medium, molar ratio of alcohol to vinyl acetate 1:2, at 40℃.100 mM cinnamyl alcohol could be 100% conversion after 90 min. It is notable that EST4 had a well tolerated in 2.5 M (335 g/L) concentration for cinnamic alcohol and was still able to maintain a high catalytic activity and the conversion was greater than 98% within 12 h. Except for cinnamic alcohol. EST4 also can tolerate alcohol concentrations greater than 2.0 M to achieve more than 97% conversion with isoamyl alcohol and citronellol. EST4 showed high transesterification activity with different structural types of primary alcohols, secondary alcohols with, is very suitable for a broad spectrum of adaptive enzyme substrate transesterification reaction has wide applicability.(5) The immobilization process of EST4 was systematically optimized to prepare the high-performance immobilized enzymes. By ammonium sulfate precipitation method EST4 lysates was purified and recovered. EST4 could be effectively recovered using 50%(w/v) ammonium sulfate and the enzyme activities recovery was higher than 98%. Different amino and epoxy resins were employed for carrier screening, and amino carrier NH was identified as the optimal carrier. Immobilized conditions are optimized by the single factor:100 mM pH 8.0 PBS buffer, at 20 ℃ 200 rpm condition, the amount of enzyme is 20 mg/g carrier and immobilized time is 3 h. Meanwhile, the synthesis of cinnamyl acetate as a model reaction was investigated the stability of the immobilized enzyme, and the results showed that the immobilized enzyme remain more than 90% residual activity for 11 consecutive batches. The stability of EST4 in the process of biocatalysis is improved and it worths in industrial application.